Abstract Biofilms and their persistence pose a serious public health concern, particularly in the hospital setting. Adhering to both biotic and abiotic surfaces, biofilms are implicated in a variety of different infections largely due to their significant decrease in antimicrobial susceptibility and clearance resistance. Not only are they found on medical devices, such as catheters, artificial joint implants, and prosthetic devices, but biofilms are also found in cystic fibrosis lungs and chronic cutaneous wounds. In the majority of bacterial species, the highly ubiquitous and important second messenger, c-di-GMP, is a central regulator of biofilm formation. We and others have recently discovered that c-di-GMP directly interacts with a subset of transcription factors belonging to the widespread NtrC-like bacterial enhancer binding protein (EBP) family to modulate biofilm gene expression as well as virulence factor, quorum sensing, and motility gene expression. In Vibrio cholerae, the causative agent of the life-threatening disease cholera responsible for 5 million cases and over 100,000 deaths per year, the response regulator VpsR is the master EBP that interacts with c-di-GMP to positively regulate biofilm gene expression in vivo in part at the vpsL biofilm gene promoter. Although EBPs typically activate RNA polymerase (RNAP) containing the alternate s factor, s54, substitutions at crucial residues in VpsR needed for EBP function have suggested that the mechanism of VpsR activation is novel. Furthermore, the mechanism by which c-di-GMP interacts with transcriptional activators to directly alter gene expression is unknown. Using V. cholerae as a model to study c-di-GMP signaling and biofilm formation in vitro and in the bacterial cell, the goal of the proposed research is to elucidate this c-di-GMP-dependent transcription mechanism. With my preliminary work, I have established an in vitro system to show for the very first time that not only can an EBP together with c-di-GMP directly activate transcription in vitro, but also VpsR together with c-di-GMP activates transcription from RNAP containing the primary s, s70. Using these conditions, I will determine in Aim 1 the specific step by which VpsR/c-di-GMP activates transcription by assessing DNA binding, RNAP recruitment, open complex formation, and promoter clearance. In Aim 2, I will use genetic and biochemical tools to investigate protein-DNA and protein-protein interactions to construct a 3-dimensional molecular map of the transcription complex. Finally, in Aim 3, I will determine the VpsR/c-di-GMP binding pocket using a high- throughput genetic screen followed by in vitro confirmation for c-di-GMP binding and transcription activation and in vivo analysis for biofilm formation. The proposed research will be the first to utilize in vitro transcriptional studies to determine how c-di-GMP interacts with transcriptional regulators to directly change gene expression. This understanding will not only provide a new paradigm in c-di-GMP-dependent transcription activation and elucidate mechanistic processes that regulate biofilm formation, but also provide the foundation needed for the development of novel chemical inhibitors against V. cholerae and biofilm-based nocosomial infections. !